The precision of axon targeting of mouse olfactory sensory neurons requires the BACE1 protease

Abstract

The β-site amyloid precursor protein cleaving enzyme 1 (BACE1) is necessary to generate the Aβ peptide, which is implicated in Alzheimer's disease pathology. Studies show that the expression of BACE1 and its protease activity are tightly regulated, but the physiological function of BACE1 remains poorly understood. Recently, numerous axon guidance proteins were identified as potential substrates of BACE1. Here, we examined the consequences of loss of BACE1 function in a well-defined in vivo model system of axon guidance, mouse olfactory sensory neurons (OSNs). The BACE1 protein resides predominantly in proximal segment and the termini of OSN axons, and the expression of BACE1 inversely correlates with odor-evoked neural activity. The precision of targeting of OSN axons is disturbed in both BACE1 null and, surprisingly, in BACE1 heterozygous mice. We propose that BACE1 cleavage of axon guidance proteins is essential to maintain the connectivity of OSNs in vivo.

Figure 1. BACE1 expression in mouse olfactory epithelium and olfactory bulb is activity dependent.

(a – c) BACE1 immunoreactivity (IR) is enriched in the axon bundles (arrows) of olfactory sensory neurons (OSNs), where it is colocalized with NCAM, an axon-specific marker protein. The insets indicate that BACE1 is partially colocalized with NCAM in the axon bundles. (d) BACE1 IR is present in the olfactory bulb, with the highest level in the glomerular layer (GL), a modest level in the external plexiform layer (EPL) and absent in the mitral cell layer (MCL). (e – f) BACE1 IR is reduced in BACE1 heterozygous mice (BACE1 +/−) and absent in BACE1 homozygous knockout mice (BACE1 −/−). (g) Quantification of BACE1 IR intensity in the olfactory bulb. (h – i) In situ hybridization reveals graded levels of BACE1 mRNA in the olfactory epithelium (h) and robust staining in the mitral cell layer of the olfactory bulb (i). Unilateral naris closure in control mice reveals a marked increase in the BACE1 mRNA levels on the closed side of the olfactory epithelium (j) but not in the mitral cells of the olfactory bulb (k). Quantitation of the BACE1 protein levels in the glomeruli of the olfactory bulb (l) reveals significant increases (m) in the dorsal, medial, and lateral regions of the olfactory bulb (p < 0.001). Black bars represent closed side, and gradient bars represent the open side.

Figure 2. BACE1 IR predominantly colocalizes with pre-synaptic neuronal marker proteins in the mouse olfactory bulb.

(a – c) Colocalization of BACE1 IR and NCAM IR in glomerular layer (GL) but not in the outer nerver layer (ONL). (d) Percentage of overlapping pixels of NCAM and BACE1 in GL and ONL. (e – g) BACE1 IR overlaps with OMP IR, a specific marker of OSNs, in GL. (h) Percentage of overlapping pixels of OMP and BACE1. (i – k) BACE1 IR overlaps with GAD65 IR, a specific marker of inhibitory periglomerular cells in GL. (l) Percentage of overlapping pixels of of GAD65 and BACE1 revealed that it is higher in GL than that in external plexiform layer (EPL). (m – o) Colocalization of BACE1 IR with MAP2 (a dendritic marker protein) immunoreactivity in GL and OPL. (p) Quantification of the overlapping level of MAP2 and BACE1. (q – s) BACE1 colocalizes with postsynaptic protein PSD95 in GL and EPL. (t) Quantification of the overlapping level of PSD95 and BACE1. * = p < 0.05.

Figure 3. Axon targeting of OSNs is altered in BACE1-deficient mice.

(a) Immunostaining using an antibody against the OR M71 revealed that M71-expressing OSN axon fibers converge to one specific glomerulus in each half bulb in wild type (BACE1 +/+) mice as indicated by the arrow. In BACE1 heterozygous (BACE1 +/−) mice (b) and BACE1 null mice (BACE1 −/−) (c), M71-expressing OSN axons project more glomeruli than one glomeruli per half bulb as indicated by the white arrowheads. (d) Quantification of the number of glomeruli receiving M71 projections per half bulb in BACE1 +/+, BACE1 +/− and BACE1 −/− mice. Differences between BACE1 +/− and BACE1 +/+, and between BACE1 −/− and BACE1 +/+ mice are statistically significant. Immunostaining using an antibody against GFP in P2-IRES-GFP transgenic animals crossed to BACE1 +/+ mice (e), BACE1 +/− mice (f) or BACE1 −/− mice (g) revealed a disturbance of axon targeting of P2-expressing OSNs in the BACE1 mutants. Quantification of the number of glomeruli receiving P2 OSN axon projections per half bulb (h) revealed statistically significant differences between BACE1 +/− and BACE1 +/+; and BACE1 −/− and BACE1 +/+ mice. Immunostaining using an antibody against MOR28 revealed that the axons of MOR28-expressing OSNs converge to one specific glomerulus in each half bulb of the BACE1 mutants (i – k). Quantification of the number of glomeruli receiving MOR28-expressing OSN axon projections per half bulb (l) revealed no statistically significant difference between BACE1 +/− and BACE1 +/+, or BACE1 −/− and BACE1 +/+ mice.

Figure 4. Increased presence of APP and APLP2 in olfactory bulbs of BACE1 heterozygous and null mice.

Representative images of immunostaining for APP in olfactory bulbs from littermate wild type (a), BACE1 +/− (b), and BACE1 −/− (c). Quantification of the intensity of immunostaining (d) revealed a significant difference between the BACE1 −/− with either the wild type or BACE 1 +/− mice (n = 3 sets of 3 littermates). A regional analysis of immunostaining (e) in the glomeruli of the olfactory bulb reveals significant increases in APP staining in the ventral and lateral regions. Representative images of immunostaining for APLP2 in olfactory bulbs from littermate wild type (f), BACE1 +/− (g), and BACE1 −/− (h). Quantification of the intensity of immunostaining (i) revealed a significant difference between the BACE1 −/− with either the wild type or BACE 1 +/− mice (n = 3 sets of 3 littermates). A regional analysis of immunostaining (j) in the glomeruli of the olfactory bulb reveals significant increases in APLP2 staining in all 4 regions. * = p < 0.05; ** = p < 0.01.

Figure 5. Reduced tyrosine hydroxylase induction in BACE1 null mice.

Tyrosine hydroxylase (TH) levels correlate with OSN input into glomeruli. Representative images of olfactory bulbs immunostained with an antibody recognizing TH (red) and a nuclear stain DAPI (blue) from a control BACE1 +/+ mouse (a), a BACE1 +/− mouse (b), and a BACE1 −/− mouse (c). Quantitation of the intensity of TH staining in glomeruli of BACE1 deficient mice (n = 4) and wild type littermate mice (n = 4) reveals a statistically significant difference (p < 0.05) between BACE1 +/+ and BACE1 −/− mice (d).

Figure 6. No significant loss of specific OSN populations or altered rates of neurogenesis in BACE1 null and heterozygous mice.

Normalized counts of genetically defined subpopulations of M71- (a), MOR28- (b), or P2- (c) expressing OSNs in a systematic series of sections spanning the entire olfactory epithelium are not significantly different (p > 0.05, mutants vs. control, n = 4). (d) Quantitation of the normalized density of BrdU-positive OSNs in a series of sections spanning the entire olfactory epithelium in BACE1 −/−, BACE1 +/− and wild type control mice reveals no significant difference (p > 0.05, mutants vs. control, n = 4).